1. Field of the Invention
This invention relates to a method and apparatus for rapidly producing constituent amino acids from proteins and peptides and, in particular, for microwave hydrolysis of proteins and peptides for amino acid analysis.
2. Description of the Related Art
There is a great need in modern biology to understand and explain the physical structure of proteins and peptides. A protein is a complex union of amino acids. A peptide is a combination of amino acids formed by the linkage of amino groups of some amino acids with carboxyl groups of other amino acids. For the purpose of this disclosure, the term "protein(s)" is intended and defined to include peptide(s).
One way to learn about proteins is to break apart or reduce the proteins to their constituent amino acids and then analyze the resulting amino acids. Proteins can be broken apart by hydrolyzing them. That is, proteins are caused to react with water such that parts of the proteins combine with a hydrogen ion (H.sup.+) and other parts of the protein combine with an hydroxyl anion (OH.sup.-) to form the constituent amino acids of the proteins.
Hirs et al. discloses the hydrolysis of proteins by heating them in sealed glass tubes with 6 N hydrochloric acid at a temperature of about 110 degrees Celcius (.degree. C.). See Hirs et al., J. Biol. Chem., 211:941-950 (1954). This process requires between 16 and 24 hours for complete hydrolysis to occur depending on the protein, since some proteins are more difficult to hydrolyze than others. Complete hydrolysis of the protein sample is necessary to fully analyze the resulting amino acids. After hydrolysis, the tube is cooled, opened and the acid evaporated forming a solid comprising the constituent amino acids of the protein. Then the solid is dissolved in a buffer solution resulting in a mixture called a hydrolysate. Analysis of a hydrolysate on a modern amino acid analyzer takes from 30 minutes to 60 minutes depending on the analyzer. The most significant rate-limiting step in obtaining amino acid data from a protein sample is, thus, the hydrolysis procedure.
Various protein hydrolysis methods have been published using mixed hydrochloric and trifluoroacetic acids (see Tsugita et al., Eur. J. Volume 124, pages 585-588, 1982) and mixed and proprionic acids (see Westall et al., Analyt. Biochem., Volume 61, pages 610-613, 1974). These methods have not resluted in widespread use.
Shui-Tein Chen et al. in "Rapid Hydrolysis of Proteins and Peptides by Means of Microwave Technology and its Application to Amino Acid Analysis", Int. J. Peptide Protein Res., Volume 30, pages 572-576, 1987 disclose that proteins were hydrolyzed in a specifically constructed Teflon.RTM. vial by heating the sample in a microwave oven. Chen et al. tried to hydrolyze proteins in Pyrex.RTM. glass tubes with 6 N hydrochloric acid but an explosion of the reaction tubes resulted inside the microwave oven due to the high pressure and temperature induced in the sealed tubes.
There is a disadvantage to hydrolyzing proteins in a Teflon.RTM. vial as disclosed in Chen et al. It is much harder to remove the resulting amino acids from a Teflon.RTM. vial than a glass one. In fact, amino acids migrate into the Teflon.RTM. vial during hydrolysis. Whereas, they don't migrate into glass during hydrolysis. This migration effect becomes more significant the smaller the sample of proteins being hydrolyzed and the larger the interior surface area of the vial.
Chen et al. disclosed hydrolysis of native ribonuclease A, oxidized ribonuclease A, and insulin B after exposing them to about 560 watts of microwave energy for 1 to 7 minutes. There are many proteins that are harder to hydrolyze than those proteins. Further, the constituent amino acids of the proteins hydrolyzed by Chen et al. do not include methionine, one of the most labile (or unstable) amino acids. Chen et al. do not disclose the specific amounts of proteins used or the percent of the hydrolyzed proteins that they recovered in the analysis.
Chen et al. discloses using vials with a volume of 2 milliliters (mL). Tsugita et al., supra., disclose hydrolyzing samples in 0.05 to 3.00 milliliters of total solution volume.
Clearly if a process and apparatus could be developed that significantly reduced the hydrolysis time of proteins, it would greatly facilitate amino acid analysis and, thus, the understanding of proteins.
An object of this invention is to provide a reliable reproducible process and apparatus for rapid hydrolysis of proteins for amino acid analysis.
Another object of this invention is to provide a reliable reproducible process and apparatus for rapid hydrolysis of microgram quantities of proteins.
Another object of this invention is to provide a reliable reproducible process and apparatus for rapid hydrolysis of proteins with greater than 60% recovery of the constituent amino acids including labile amino acids, such as serine, methionine and threonine.